JP6990814B2 - Duck plague virus and its use - Google Patents

Description

The present invention relates to novel viruses and their use. More specifically, the invention relates to novel Duck Enteritis Virus constructs and their use for expressing or delivering a polypeptide of interest to animals, particularly poultry. The present invention is particularly suitable for vaccination of poultry against avian pathogens.

Poultry meat and eggs are important food sources, and their consumption is constantly growing due to the growing human population and the balance between high quality and price. Due to the recent epidemic of bird flu, public interest has focused on poultry health as well as food safety and security, and poultry vaccine technology has become a global concern.

Recombinant viruses that express pathogen proteins are commonly used as poultry vaccines against target pathogens. Vaccines containing such viruses can induce the expression of foreign pathogen proteins or fragments thereof in infected cells, thereby inducing specific defensive humoral immunity as well as cell-mediated immunity.

In this regard, several viruses that incorporate foreign genes derived from pathogens have been developed for use as viral vector vaccines. These viral vectors (or recombinant viruses) are typically tripoxviruses such as fowlpox (European Patent Application Publication No. 0,517,292), herpesviruses, especially HVT (eg, International Publication No. 87/04463,). It is based on US Pat. No. 5,980,906), US Pat. No. 5,853,733), Newcastle's disease virus (NDV) or triadenovirus. These recombinant avian viruses exhibit variable levels of protection depending on the disease and / or animal.

For example, poxviruses, NDVs, and adenoviruses do not persist in chickens and are not considered the best candidates for long-lasting immunity in chickens. Recombinant HVTs that express the antigen have shown advantages and are currently commercialized for vaccination in chickens (eg, Vectormune® IBD, Vectormune® ND, or Vectormune®. ) LT).

However, given the increasing number and variety of pathogens and the ever-increasing poultry consumption, there is a need for alternative vaccination plans and / or systems that can be used to elicit effective protective immunity in poultry. exist. In particular, there is a need for an effective system to acquire immunity in very young animals (within 3 days) or in ovo.

In this regard, new viral cellotypes have been investigated with the aim of finding alternative compatible viral vectors that improve vaccination in animals, especially in poultry, and enable stable protein expression and effective defense. rice field.

WO 2014/0036735 discusses the possible use of the duck enteritis virus in chickens. DEV naturally infects ducks or geese, but there is no known tropism for chickens. This document suggests that the DEV construct can be administered to 1 week old chickens by intramuscular injection. However, only late doses are reported in this document.

However, by further experimenting with DEVs, the inventors found that such viruses were lethal when administered to young chickens (within 3 days) or in ovo. Surprisingly, it is well tolerated to administer wild-type DEVs (or DEV constructs containing all natural genes proposed in WO 2014/0036735) to chickens one week after hatching. As it seems, administration of such constructs on the first day after hatching or in ovo causes mass mortality of the animal (ie, between 80-100%). Even more surprising, the inventors can use it in poultry, including very early stages (within 3 days) or in ovo, which can cause substantial and early stage protein expression in vivo. It was possible to modify the structure of a DEV to create a possible DEV construct. Therefore, such viruses represent a novel and potent vector for vaccination of poultry.

European Patent Application Publication No. 0,517,292 International Publication No. 87/04463 U.S. Pat. No. 5,980,906 U.S. Pat. No. 5,853,733 International Publication No. 2014/0036735 U.S. Pat. No. 6,764,684 U.S. Pat. No. 6,866,852

Molecular Cloning: A Laboratory Manual. 4th Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 2012

The present invention is suitable for expressing a gene or protein in vivo in poultry, including in animals, especially in the very early stages (ie, 3 days after hatching or earlier, and in ovo). Provide a new virus construct. In particular, the invention provides novel DEVs obtained by inactivating the US4 and US5 genes, which are (i) attenuated in vivo, especially in chickens, (ii). It demonstrates that it is stable and can express foreign genes in a manner suitable for inducing defensive immunity. In addition, these defective attenuated DEVs maintain a fast growth rate, allowing the production of high titers. Such DEVs, for example, have no known natural tropism for chickens, so even if such DEV constructs are used to vaccinate chickens, they are transferred or contaminated to unvaccinated animals. There is no risk of. In addition, chickens do not have transitional antibodies or immunity to DEV and the viruses of the invention can be used to induce very early immunization in vaccinated animals. Surprisingly, as previously shown, wild-type DEVs are lethal in young chickens or in ovo, whereas the DEVs of the invention are safe and effectively express the gene of interest in vivo. It is possible. Thus, such novel DEVs represent a very potent vector for vaccination of non-human animals, especially poultry, and for conferring early protective immunity.

More specifically, the subject of the present invention relates to the duck enteritis virus (DEV), in which the virus has the inactive US4 and US5 genes. According to the present invention, it is possible to obtain a viable and stable replicating DEV by inactivating these genes, and if such a virus is used, a recombinant DEV can be obtained by inserting a foreign gene material. It actually reveals what can be created. The results further reveal that such foreign genetic material is highly expressed from such viruses upon cell infection, and such expression remains stable over time. Furthermore, and notably, natural DEVs and many other defective DEV constructs created by the inventors are pathogenic (3 days after hatching or earlier) and in young chickens in ovo. Although found to be or lethal, inactivating US4 and US5 creates an attenuated virus that can be safely used to express proteins and antigens in young chickens, including in ovo. Will be done. Such findings are quite surprising and offer great benefits and usefulness to the virus.

Therefore, a further subject of the invention relates to a duck enteritis virus (DEV) in which the virus has the Inactive US4 and US5 genes and contains foreign nucleic acids.

According to certain embodiments, the US4 and US5 genes are mutated, deleted, or disrupted; and / or foreign nucleic acids replace all or part of the US4 and US5 gene sequences. And / or foreign nucleic acids encode avian pathogens.

In a further specific embodiment, the DEV of the invention further comprises an inactive UL4, UL23, or US7 gene.

A further subject of the invention is a nucleic acid molecule containing the genome of a DEV with the inactive US4 and US5 genes.

The invention further relates to host cells containing the DEVs or nucleic acids defined above.

The present invention is a method for producing or replicating a DEV defined above, comprising infecting competent cells with a nucleic acid molecule or a DEV defined above and collecting the DEV. Also provide.

The present invention also relates to methods for making recombinant DEVs, comprising inserting a foreign nucleic acid on behalf of all or at least 20% of the US4 and US5 gene sequences.

The invention also provides a DEV, nucleic acid, or host cell as defined above, a pharmaceutically or veterinarily acceptable excipient or carrier, and optionally a composition comprising an adjuvant.

The invention also provides DEVs, nucleic acids, or host cells as defined above, pharmaceutically or veterinarily acceptable excipients or carriers, and optionally vaccines containing adjuvants.

Further subjects of the invention are vaccination of birds, in particular poultry, more particularly chickens, and more particularly young poultry (3 days after hatching or earlier or in ovo). With respect to the compositions, DEVs, nucleic acids or host cells defined above for use in immunizing this.

A further subject of the invention is to induce protective immunity to birds, in particular poultry, more particularly chickens, and more specifically young poultry (3 days after hatching or earlier or in ovo). With respect to the compositions, DEVs, nucleic acids or host cells defined above for use in.

The present invention is a method of vaccination of non-human animals, in particular poultry, more particularly chickens, and more particularly young poultry (3 days after hatching or earlier or in ovo). It also relates to a method comprising administering the composition or virus as defined above to said non-human animal.

A particular subject of the invention is a method of vaccination of poultry, comprising in ovo administration of the composition or virus as defined above.

Another particular subject of the invention is a method of vaccination of poultry, comprising administration of the composition or virus defined above on day 1 after hatching (ie, within about 24 hours). be.

In a further aspect, the invention is a method for inducing an immunogenic or defensive response in a non-human animal against one or more avian pathogens, said non-human animal, in particular poultry, more particularly. A method comprising administering to chickens, and more particularly young poultry (3 days after hatching or earlier or in ovo), the composition, vaccine or virus as defined above. offer.

The virus or composition of the present invention may be administered by any route. Preferably, the virus or composition of the invention is administered in ovo or by subcutaneous (eg, s.c.) Injection 1 or 2 days after hatching for very early immunization.

The present invention is a vaccination kit for immunizing birds, the following components:
The composition on the effective amount, and
b. Further provided a vaccination kit comprising means for administering the composition to the birds.

The present invention may be used to express a polypeptide in any animal, preferably for vaccination of birds, and the present invention is one or several polypeptides or peptides, specifically. Is suitable for expressing immunogenic peptides of avian pathogens.

A schematic diagram of the duck plague virus (DEV) genome and the location of the US gene is shown. The position of the insertion site in the parent DEV genome and the schematic diagram of the genome structure of pUC18-KAPEVAC-US4US5del-BacVP2 and DEV / US4US5del / BacVP2 are shown. The locations of Junction 1, Junction 2, and Junction 3 used for amplification in the PCR reaction are shown. It shows VP2 expression by CEF infected with DEV / US4US5del / BacVP2 in the black plaque assay. The position of the insertion site in the parent DEV genome and the schematic diagram of the genome structures of pUC18-KAPEVAC-US4US5del and DEV / US4US5del are shown. The location of junction 1 used for amplification in the PCR reaction is shown. The position of the insertion site in the parent DEV genome and the schematic diagram of the genome structure of pUC18-KAPEVAC-UL23del-Coa5VP2 and DEV / US4US5del / UL23 / Coa5VP2 are shown. The locations of Junction 1, Junction 2, and Junction 3 used for amplification in the PCR reaction are shown. The position of the insertion site in the parent DEV genome and the schematic diagram of the genome structure of pUC18-KAPEVAC-UL26-Coa5VP2 and DEV / US4US5del / UL26 / Coa5VP2 are shown. The locations of Junction 1, Junction 2, and Junction 3 used for amplification in the PCR reaction are shown. The position of the insertion site in the parent DEV genome and the schematic diagram of the genome structure of pUC18-KAPEVAC-UL45-Coa5VP2 and DEV / US4US5del / UL45 / Coa5VP2 are shown. The locations of Junction 1, Junction 2, and Junction 3 used for amplification in the PCR reaction are shown. The position of the insertion site in the parent DEV genome and the schematic diagram of the genome structure of pUC18-KAPEVAC-UL50-Coa5VP2 and DEV / US4US5del / UL50 / Coa5VP2 are shown. The locations of Junction 1, Junction 2, and Junction 3 used for amplification in the PCR reaction are shown. It shows VP2 expression by CEF infected with DEV / US4US5del / UL23 / Coa5VP2, DEV / US4US5del / UL26 / Coa5VP2, DEV / US4US5del / UL45 / Coa5VP2, or DEV / US4US5del / UL50 / Coa5VP2 in the black plaque assay. A Western blot assay that detects VP2 protein expression by DEV / US4US5del / UL23 / Coa5VP2, DEV / US4US5del / UL26 / Coa5VP2, DEV / US4US5del / UL45 / Coa5VP2, or DEV / US4US5del / UL50 / Coa5VP2. 1: DEV / US4US5del / UL23 / Coa5VP2; 2: DEV / US4US5del / UL26 / Coa5VP2; 3: DEV / US4US5del / UL45 / Coa5VP2; 4: DEV / US4US5del / UL50 / Coa5VP2; 5: Parent DEV; 6: CEF

The present invention generally relates to attenuated DEVs containing foreign gene sequences. The present invention is used to vaccinate compositions containing such DEVs, as well as animals, in particular poultry, and more particularly young poultry (3 days after hatching or earlier or in ovo). Also related to its use.

The present disclosure will be best understood by reference to the following definitions.

The definition term "virus" specifically refers to a viral particle containing a nucleic acid molecule (eg, a genome) encapsulated in a capsid or capsule. The term "virus" also refers to a viral vector or isolated viral genome.

The term "recombination" refers to a molecule created, designed or modified using genetic technology. In connection with a virus, the term "recombinant" more specifically refers to a virus whose genome (or its ancestral genome) has been modified by the insertion or deletion of at least one nucleic acid sequence.

The term "foreign nucleic acid" refers to a nucleic acid that is not naturally found in the viral genome in the context of the virus, or is found naturally in the genome but in different forms or at different positions.

In this description, the term "nucleic acid" refers to any nucleic acid molecule or sequence such as deoxyribonucleotide (DNA) or ribonucleotide (RNA), which may be, for example, single-stranded or double-stranded. Nucleic acid may or may not contain an ORF. Nucleic acid molecules can be made by techniques known per se in the art such as artificial synthesis, recombinant techniques, enzymatic techniques, replication in host cells, or combinations thereof.

"Gene" represents a nucleic acid molecule or sequence comprising an open reading frame encoding a product such as a polypeptide (eg, peptide, protein, etc.) or RNA.

In the context of the present invention, a DEV having an "inactive" gene represents a DEV that cannot express the functional protein or RNA encoded by the gene. Thus, the Inactive US4 gene represents a US4 gene that cannot encode, mutate, delete, and / or discontinue the wild-type US4 protein. The Inactive US5 gene represents a US5 gene that cannot encode, mutate, delete, and / or discontinue the wild-type US5 protein. If the US5 gene contains the 5'US5 and 3'US5 coding sequences, the inactive US5 cannot encode any wild-type protein encoded by the 5'US5 and 3'US5 coding sequences, mutated or deleted. And / or representing the interrupted US5 gene, eg, both 5'US5 and 3'US5 contain mutations or deletions or interruptions.

As used herein, the term "attenuated" is a virus that does not cause disease in the animal model. Attenuated viruses can typically replicate in the host and do not cause host death. The attenuated virus, more specifically, represents a virus that is not pathogenic in the embryo when injected at a dose of 1 × 10 ³ plaque forming units (pfu) / egg. Highly preferred attenuated viruses are at least 70% injected eggs, even more preferably at least 80% injected eggs, and even more preferably at least 90%, 95%, 97%, 98%, 99%. Or with more injected eggs, a dose of 1 x 10 ³ pfu / egg is safe. The attenuated virus of the present invention is also safe for post-hatching injections, including day 0 (ie, between 0.1-48 hours after hatching).

The term "birds" is intended to include all types of birds, such as birds of the class of birds, namely winged, winged, bipedal, endothermal, and spawning vertebrates. ing. In the context of the present invention, birds or bird species are more specifically poultry, more preferably chickens of economic and / or agricultural interest such as chickens and turkeys, or ornamental birds such as swans and parrots. That is.

As used herein, the term "vaccine" refers to an agent that may be used to provoke, stimulate or amplify an immune response in an organism.

"Immune response" refers to the development of a cellular and / or antibody-mediated immune response to a composition or vaccine of interest in a host. Usually, an "immune response" comprises the production of antibodies, B cells, helper T cells, and / or cytotoxic T cells specifically directed to the antigen contained in the composition or vaccine of interest. Preferably, the immune response is defensive so that resistance to new infections is enhanced and / or the clinical severity of the disease is reduced.

The term "in ovo" administration or injection generally means inoculation or injection into an embryo contained in an egg. In ovo injections are preferably given at any time between the 5th and 1st days before hatching.

Duck plague virus Duck plague virus (DEV), also known as duck virus enteritis virus (DVEV), naturally infects ducks and geese. The complete nucleotide sequence of DEV has been established and is available online (see, eg, JQ673560). The viral genome contains about 162 Kb and encodes nearly 80 different proteins. Several cellotypes and strains of DEV have been isolated, such as Janssen strain, CSC strain, CHv strain, VAC strain, and 2085 strain. VAC strain: ID EU082088.2; Anatidae isolate C-KCE: ID KF263690.1; Anatidae CHv: ID JQ647509.1; Anatidae 2085: ID JF999965; Anatidae CV: ID KJ549663.1 or Anatidae Complete sequences of several DEV strains such as CSC: ID JQ673560.1 are available at Genbank.

DEV remains poorly characterized and its use as a vector to express genes has not been investigated in depth. For example, Liu et al. (2013) and WO 2014/0036735 attempted to use recombinant DEVs to express genes in chickens. They utilized DEV constructs that cloned nucleic acids between the US7 and US8 genes in the viral genome without altering natural gene expression. It has been reported that such constructs may have been transferred to 1 week old chickens by intramuscular injection, however, in this document or in any other prior art document, poultry in ovo. There is no disclosure of any possible use of DEV for vaccination or for vaccination of young poultry, i.e., 3 days or before, especially 1 or 2 days after inoculation.

By conducting further experiments with DEV, we surprisingly found that the virus was lethal when administered to young chickens (within 3 days) or in ovo. rice field. Surprisingly, administration of wild-type DEVs (or DEV constructs containing any natural gene as proposed in WO 2014/0036735) to chickens one week after hatching is well tolerated. However, as reported in Example 1, administration of such constructs 1 day after hatching or in ovo causes mass mortality of the animal (ie, between 80-100%).

Even more surprising, the inventors can use it in poultry, including very early stages (within 3 days) or in ovo, which can cause substantial and early stage protein expression in vivo. It was possible to modify the structure of a DEV to create a possible DEV construct. More specifically, we conducted further studies with DEVs to create various recombinants with different gene deletions or alterations. Surprisingly, by inactivating both the US4 and US5 genes, we are (i) attenuated in vivo, especially in chickens, and (ii) stable and defensive immunity. We have found that it is possible to obtain recombinant DEVs capable of expressing foreign genes in a manner suitable for induction. The results further show that such foreign genetic material is highly expressed from such viruses upon cell infection and such expression remains stable over time. Furthermore, and notably, inactivation of US4 and US5 creates attenuated DEVs that can be safely used to express proteins or antigens in young poultry and in ovo. DEVs with only the inactive US4 gene or only the inactivated US5 gene are still pathogenic or lethal in young poultry (less than 3 days old or in ovo). DEVs, for example, have no known natural tropism for chickens, so even if the DEV constructs of the invention are used for chicken vaccination, they also include the risk of transfer or contamination to unvaccinated animals. do not have. In addition, chickens do not have transitional antibodies or immunity to DEV and the viruses of the invention can be used to induce a very early start of immunity in vaccinated animals.

Therefore, the subject of the present invention relates to a duck enteritis virus (DEV) in which the virus has the inactive US4 and US5 genes.

A further subject of the invention relates to a duck enteritis virus (DEV) in which the virus has the inactive US4 and US5 genes and contains foreign nucleic acids.

The DEV of the present invention may be prepared from any DEV species or strain. Janssen shares, VAC shares (ID EU082088.2), C-KCE shares (ID KF263690.1), CHv shares (ID JQ647509.1), 2085 shares (ID JF999965), CV shares (ID KJ549663.1) or CSC shares Several strains of DEV, such as (ID JQ673560.1), have been reported and are available from published collections.

In a preferred embodiment, the DEV of the invention is at least 90% sequence identity to the Janssen strain or CSC strain, or Janssen strain or CSC strain, more preferably at least 95%, at least 96%, at least 97%, at least 98%. Alternatively, it is derived from or prepared from a parent strain selected from any DEV strain having at least 99% sequence identity.

The present invention is safe when injected into in ovo or young poultry by inactivating (ie, inactivating or deleting) the US4 and US5 genes and replicates in poultry to express the protein. It is revealed that it is possible to create an attenuated DEV that can be used. In particular, as shown in the Examples, the DEV construct with the inert US4 and US5 genes is stable, replicable in culture and can be safely administered to poultry eggs or young poultry. Thus, such viruses can be used to create recombinant DEVs containing foreign nucleic acid materials, especially antigenic coding genes, to express such genes in poultry.

In the context of the present invention, a DEV having an inactive gene represents a DEV in which neither the functional protein encoded by the gene nor RNA can be expressed. Thus, the Inactive gene specifically represents a gene that cannot encode a wild-type protein, is mutated, is deleted, and / or is interrupted.

In certain embodiments, the gene is inactive as a result of one or several mutations in the coding sequence, particularly point mutations in the coding sequence that interfere with the expression of the full-length protein. Such mutations can introduce arrests or nonsense codons into the sequence, or cause substitutions of essential amino acid residues in the encoded protein, resulting in an inactive protein.

In another embodiment, at least a portion of the (coding) sequence of the gene, more particularly at least 20%, more preferably at least 30%, at least 40%, at least 50% of the (coding) sequence of the gene. The gene is inactive as a result of deletions of at least 60%, at least 70%, at least 80%, or at least 85%, up to 100%. In a preferred example, the DEVs of the invention have a deletion of at least 300 bp of (the coding sequence for) the inactivated gene. Such deletions remove the coding sequence and thus interfere with expression of wild-type proteins, or even any protein.

In this regard, a particular embodiment of the invention relates to a duck enteritis virus (DEV) in which the virus has a deletion in / of the US4 and US5 genes, particularly in the coding sequence of the US4 and US5 genes.

The US4 and US5 genes in DEV are expected to encode proteins. However, the actual function of these genes remains unclear. Until the present invention, the ability to produce a DEV virus with a US4-US5 double deficiency has not been known, and such a DEV virus has no ability to replicate and express foreign genes in birds without lethality. It was unknown.

The US4 gene is typically composed of 1380 bp of the DEV genome and encodes a protein containing approximately 459 amino acid residues. US4 is highly conserved among DEV strains. With reference to the CSC strain, the US4 gene corresponds to the genomes nt141123 through nt142502. It is understood by those skilled in the art that the exact location of the US4 gene can be easily identified in any DEV strain using the information contained in this application and commonly known facts, or by sequence alignment. In a particular DEV of the invention, at least 20%, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the US4 (coding) gene sequence. , Up to 100% are deleted. In a preferred example, the DEVs of the invention have at least 500 bp, more preferably at least 600, 700, 800, 900, 1000, or more deletions of the US4 (coding) gene sequence. In certain embodiments, the DEVs of the invention have deletions of the US4 gene sequence spanning at least nt200-1000, more preferably at least nt150-1150, and even more preferably at least nt100-1300. In certain examples, the DEVs of the invention have a deletion of the US4 gene sequence from nt51 to nt1330 (ie, greater than 90%). In another particular embodiment, the DEVs of the invention have deletions of all (nt1-1380) of the US4 gene sequence.

The US5 gene of DEV encodes a glycoprotein and its function remains unknown. In most DEV strains (eg, VAC, CSC, C-KCE, CHv, CV), the US5 gene is composed at approximately 1620 bp and encodes a protein with approximately 539 amino acid residues. In some DEV strains, such as the 2085 and Janssen (or Kapevac) strains, the US5 gene is separated by a small intergenic region of approximately 25 bp, two relatively short coding regions, approximately 396 bp of 5'US5. And contains approximately 1197 bp of 3'US5 (see Figure 1). With reference to the CSC strain, the US5 gene corresponds to the genomes nt142662 to nt144281. It is understood by those skilled in the art that the exact location of the US5 gene can be easily identified in any DEV strain using the information contained in this application and commonly known facts, or by sequence alignment. In a particular DEV of the invention, at least 20%, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the US5 (coding) gene sequence. , Up to 100% are deleted. In a preferred example, the DEVs of the invention have at least 500 bp, more preferably at least 600, 700, 800, 900, 1000, or more deletions of the US5 (coding) gene sequence. In certain embodiments, the DEVs of the invention have deletions of the US5 gene sequence spanning at least nt200-1000, more preferably at least nt100-1100, and even more preferably at least nt80-1120. If the US5 gene contains two ORFs, it is preferred that both ORFs are inactive. In this regard, if the DEV is prepared from a virus strain having a US5 gene sequence containing a single ORF, the DEV is preferably at least 90% of the ORF, such as a deletion of at least nt51 to nt1570 of the US5 gene sequence. Deletions, and more particularly all deletions of the US5 gene. When the DEV is prepared from a virus strain having a US5 gene sequence containing two ORFs, the DEV is preferably deleted by at least 90% of each of the ORFs, more preferably at least 90% of the first ORF. , A complete intergenic region, and a deletion containing at least 90% of the second ORF.

In certain embodiments, the DEVs of the invention have deletions of at least a portion of the US4 gene, an entire US4-US5 intergenic region, and a contiguous region spanning a portion of the US5 gene.

In a further specific embodiment, the DEV comprises a deletion of at least 50% of the US4 gene, all of the intergenic region between the US4 and US5 genes, and a nucleotide region containing at least 50% of the US5 gene.

In a more preferred embodiment, the DEV comprises deletion of the entire US4 gene, the entire intergenic region between the US4 and US5 genes, and the nucleotide region containing all of the US5 genes. A specific example of such a structure is, for example, DEV / US4US5 / BacVP2.

As shown above, the DEVs of the invention can contain one or several foreign nucleic acids of interest. Foreign nucleic acids are generally under the control of transcriptional promoters. Preferably, the promoter is cloned with the foreign nucleic acid. The promoter may be any natural or synthetic promoter derived from a cellular or viral gene. Examples of suitable promoters include, for example, the chicken beta actin (Bac) promoter such as Coa5 or a derivative thereof, the Pec promoter, the mouse cytomegalovirus (Mcmv) earliest (ie) 1 promoter, the human cytomegalovirus promoter (Hcmv), Includes the Simian virus (SV) 40 promoter, and the Raus sarcoma virus (RSV) promoter, or any fragment thereof that retains promoter activity. In the variant, the foreign nucleic acid is cloned downstream of the transcriptional promoter present in the DEV genome and is under its transcriptional control.

In certain embodiments, the foreign nucleic acid is the DEV virus in addition to the existing US4 gene sequence (thus deactivating the gene by interrupting the gene sequence) or by replacing the deleted sequence of the US4 gene. It is located in the US4 gene sequence of the genome or in the mutant US4 gene sequence. In another embodiment, the foreign nucleic acid is the DEV virus in addition to the existing US5 gene sequence (thus inactivating the gene by interrupting the gene sequence) or by replacing the deleted sequence of the US5 gene. It is located in the US5 gene sequence of the genome or in the mutant US5 gene sequence.

In a preferred embodiment, the DEV of the invention has a deletion in the US4 and US5 gene sequences and contains a foreign nucleic acid located in place of the deleted nucleotide.

In another embodiment, the DEV of the invention has the inactive US4 and US5 genes, the UL4 gene, the UL44 gene, the UL27-UL26 intergenic region, the UL23 gene, the UL45-UL46 intergenic region, and the UL50-UL51 intergenic region. It contains foreign nucleic acids located at different cloning sites, such as a region, a US7 gene, a US7-US8 intergenic region, or a site selected from the US10 gene. In this case, the foreign nucleic acid may be cloned in place of all or part of the gene, or may be inserted into the gene.

In addition, the DEVs of the invention can contain several foreign nucleic acids. In this regard, several foreign nucleic acids may be inserted at the same location in the virus, eg, in the US4 / US5 region described above, under the control of a single or several different promoters. Instead, the foreign nucleic acid typically replaces all or part of the endogenous gene or region at the cloning site in the US4 / US5 region described above, and preferably the UL4 gene, UL44 gene, UL27-. At least one cloning site in a different region selected from the UL26 intergenic region, UL23 gene, UL45-UL46 intergenic region, UL50-UL51 intergenic region, US7 gene, US7-US8 intergenic region, or US10 gene, etc. It may be inserted into different cloning sites of the virus.

The UL4 gene is typically composed of 717 bp of the DEV genome. With reference to the CSC strain, the UL4 gene corresponds to the genomes nt112845 to nt113561. It is understood by those skilled in the art that the exact location of the UL4 gene can be easily identified in any DEV strain using the information contained in this application and commonly known facts, or by sequence alignment. In certain embodiments, the present invention relates to a duck enteritis virus (DEV) in which the virus has the inactive US4, US5 and UL4 genes. More specifically, the invention has the US4, US5 and UL4 genes in which the virus is inactive and has been cloned into the US4 / US5 gene or by replacing the UL4 gene, preferably at least 20% of the gene. It relates to a DEV containing the first foreign nucleic acid. In a particular DEV of the invention, at least 20%, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100% of the UL4 gene sequence. Is missing. In a preferred example, the DEVs of the invention have at least 500 bp of the UL4 gene sequence, more preferably at least 600, 700, 800, 900, 1000, or more deletions.

The UL23 gene is typically composed of 1077 bp of the DEV genome. With reference to the CSC strain, the UL23 gene corresponds to the genomes nt77997 to nt79073. It is understood by those skilled in the art that the exact location of the UL23 gene can be easily identified in any DEV strain using the information contained in this application and commonly known facts, or by sequence alignment. In certain embodiments, the present invention relates to a duck enteritis virus (DEV) in which the virus has the inactive US4, US5 and UL23 genes. More specifically, the invention has the US4, US5 and UL23 genes in which the virus is inactive and has been cloned into the US4 / US5 gene or by replacing the UL23 gene, preferably at least 20% of the gene. It relates to a DEV containing the first foreign nucleic acid. In a particular DEV of the invention, at least 20%, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100% of the UL23 gene sequence. Is missing. In a preferred example, the DEV of the invention has at least 500 bp of the UL23 gene sequence, more preferably at least 600, 700, 800, 900, 1000, or more deletions. In certain embodiments, the DEVs of the invention have deletions of the UL23 gene sequence spanning at least nt200-900, more preferably at least nt100-1000, and even more preferably at least nt80-1000. In certain examples, the DEVs of the invention have a deletion of nt51 to nt1027 (ie, about 90%) of the UL23 gene sequence.

The US7 gene is typically composed of 1116 bp of the DEV genome. With reference to the CSC strain, the US7 gene corresponds to the genomes nt145769 to nt146884. It is understood by those skilled in the art that the exact location of the US7 gene can be easily identified in any DEV strain using the information contained in this application and commonly known facts, or by sequence alignment. In certain embodiments, the present invention relates to a duck enteritis virus (DEV) in which the virus has the inactive US4, US5 and US7 genes. More specifically, the invention has the US4, US5 and US7 genes in which the virus is inactive and has been cloned into the US4 / US5 gene or by replacing the US7 gene, preferably at least 20% of the gene. Concerning a DEV containing a first foreign nucleic acid. In a particular DEV of the invention, at least 20%, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100% of the US7 gene sequence. Is missing. In a preferred example, the DEVs of the invention have at least 500 bp of the US7 gene sequence, more preferably at least 600, 700, 800, 900, 1000, or more deletions.

The present invention was cloned into a first foreign nucleic acid in which the virus was cloned into the US4 / US5 gene and an intergenic region located between the UL27 and UL26 genes, preferably with at least 20% of the gene replaced. It is also involved in DEVs containing a second foreign nucleic acid. The present invention also relates to a DEV in which the virus comprises the inactive US4 and US5 genes and the virus comprises a foreign nucleic acid cloned into an intergenic region located between the UL27 and UL26 genes. With reference to the CSC strain, the intergenic region located between UL27 and UL26 corresponds to the genome from nt72195 to nt72646. Cloning may be performed at any location in such a main, more preferably between nt72300 and nt72500, even more preferably between nt72350 and nt72450. In certain embodiments, cloning is performed between nt72431 and nt72432.

The present invention contains the UL4 gene in which the virus is inactive and the virus is cloned into an intergenic region located between the US7 and US8 genes, or between the UL45 and UL46 genes, or between the UL50 and UL51 genes. It also relates to DEVs containing foreign nucleic acids. With reference to the CSC strain, the intergenic region located between UL45 and UL46 corresponds to nt25132 to nt25352 of the genome. Cloning may be performed at any of the main locations, such as between nt25200 and nt25300, more preferably. In certain embodiments, cloning is performed between nt25275 and nt25276. With reference to the CSC strain, the intergenic region located between UL50 and UL51 corresponds to the genome from nt15914 to nt16063. Cloning may be performed at any location in the main, such as between nt15970 and nt16010, more preferably. In certain embodiments, cloning is performed between nt15979 and nt15980.

Virus construction and cloning can be accomplished by techniques known per se in the art. Gene cloning and plasmid construction are well known to those of skill in the art and are standard molecular biology techniques (Molecular Cloning: A Laboratory Manual. 4th Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 2012). Can be implemented essentially by. Typically, a recombinant virus is homologous recombination between the viral genome and a construct (eg, a homologous plasmid) containing the nucleic acid to be inserted, with nucleotides flanking from the insertion site to allow recombination. May be prepared by. Cloning can be performed with or without deletion of the endogenous sequence. In certain embodiments, the recombinant sequence is cloned by replacing at least a portion of the genomic sequence, such as at least 50 nucleotides or more. Such deletions increase the ability of the virus to clone.

In the construction, the sequence containing the targeted insertion region is typically first cloned into a suitable vector to create a homologous vector. Examples of vectors include plasmids such as pBR322, pBR325, pBR327, pBR328, pUC18, pUC19, pUC7, pUC8, or pUC9; phages such as lambda phage and M13 phage; or cosmids such as pHC79. The target region sequence is incorporated into the vector by a conventional cloning method. The target region sequence used is preferably long enough to allow subsequent in vivo homologous recombination with the DEV viral genome. Preferably, the cloned target region sequence has a nucleotide length greater than 300, at least approximately 100 nucleotides in length, typically between 500 and 2000 nucleotides. The foreign nucleic acid (typically containing the gene and promoter) is then inserted into the target region cloned into the vector. Preferably, a portion of the sequence of the target region is on each side of the inserted insert long enough to allow homologous recombination (eg, at least 50 nucleotides, preferably at least 100 nucleotides). The insertion shall be made so that it remains. Foreign nucleic acids can be introduced into the target region cloned by classical techniques such as restriction enzymes and ligation techniques. Where appropriate, mutations may be introduced at specific sites in the target area to create new cleavage sites for restriction enzymes. Conventional mutagenesis methods well known to those of skill in the art, such as in vitro mutagenesis or PCR, may be used for that purpose. The homologous vector in which the foreign nucleic acid is inserted into the target region is then subjected to DEV-infected cells or DEV genomic trans using known techniques such as electroporation, calcium phosphate, lipofectin-based methods, or similar methods. It may be introduced into fect cells. Thereby, the recombinant virus is produced by recombination between the virus and the vector in the cell. The resulting recombinant virus is a functional assay that uses known techniques to detect any product encoded by, for example, hybridization, sequencing, PCR or foreign nucleic acid, as described in the Examples. Can be genotypically or phenotypically selected. The selected recombinant virus can be cultured on a large scale in cell culture and then the recombinant virus can be collected.

Foreign Gene The DEV of the present invention can contain any foreign nucleic acid, preferably any foreign gene. The foreign gene can encode any product of interest, such as RNA or a biologically active and / or immunogenic (eg, antigenic) protein, polypeptide or peptide. In a preferred embodiment, the foreign gene encodes a peptide or polypeptide derived from an antigen, and even more preferably an antigen of a pathogenic organism that can cause infection in animals, especially birds. Examples of pathogens that cause infections in birds include viruses, bacteria, fungi, protozoans, and the like. The immunogenic (poly) peptide may preferably be (derived from) a surface protein, secretory protein, or structural protein of the pathogen, or a fragment thereof. The polypeptide can be derived, for example, from any source of virus, prokaryote, eukaryote or synthesis.

In a preferred embodiment, the foreign gene encodes an antigenic peptide of a bird pathogen.

Specific examples of pathogens are avian influenza virus, avian paramyxovirus type 1, which is also called Newcastle's disease virus (NDV), avian mycoplasma virus, Marex's disease virus, Gumboro disease virus, which is Infectious bursal tracheitis virus (ILTV), infectious bronchitis virus (IBV), Escherichia coli, Salmonella spp., Pasteurella multocida, also known as infectious bursal disease virus (IBDV), Riemerella anatipestifer, Ornithobacterium rhinotracheale, Mycoplasma gallisepticum, Mycoplasma gallisepticum, Mycoplasma synoviae, or mycoplasma that infects avian species. Include without.

Priority, foreign genes are NDV F protein, NDV HN protein, IBDV VP2 protein, ILTV gB protein, mycoplasma galicepticum 40K protein, or avian influenza virus surface protein hemagglutinin (HA), or its immunity. Encodes an antigen selected from the progenitor fragment. In the context of the present invention, the term protein "fragment" preferably represents a fragment containing at least 5 consecutive amino acid residues, even more preferably 5-100 amino acid residues of said protein. In a preferred embodiment, such fragments contain at least one epitope and / or are immunogenic in vivo, i.e., capable of causing the production of antibodies that bind to full-length proteins.

Specific examples of immunogenic peptides include, for example, peptides containing 1-453 of VP2, 1-469 of gB, or 1-540 amino acid residues of F.

Preferred DEV
Preferred DEVs of the invention include deletions of all US4 and US5 genes.

Specific DEVs of the invention include deletions of at least 50% of the nucleotide sequence of the US4 gene sequence, the entire US4-US5 intergenic region, and the proximity region containing at least 50% of the nucleotide sequence of the US5 gene sequence.

Further specific DEVs of the invention include deletions of the entire nucleotide sequence of the US4 gene sequence, the entire US4-US5 intergenic region, and the near region containing all of the nucleotide sequences of the US5 gene sequence.

In the preferred DEV of the invention, the foreign nucleic acid encodes an avian antigen, more preferably a VP2, HN or F protein or an immunogenic fragment thereof.

Another preferred DEV of the invention is the inactive US4 and US5 genes as well as the deletion of at least nt100-nt1000 in the US7 gene sequence, the deletion of at least nt100-nt1200 in the UL4 gene sequence, and / or the deletion of the UL23 gene sequence. Includes at least one additional deletion selected from deletions of at least nt100-nt1000.

Another preferred DEV of the invention is the inactive US4 and US5 genes, and preferably the UL4 gene, UL44 gene, UL27-UL26 intergenic region, UL23 gene, UL45-UL46 intergenic region, UL50-UL51 intergenic region. , US7 gene, US7-US8 intergenic region, or at least one foreign nucleic acid cloned into a different region selected from the US10 gene.

Nucleic Acids The present invention also relates to nucleic acid molecules containing the genome of a DEV with the inactive US4 and US5 genes. Such nucleic acids may be single-stranded or double-stranded, DNA or RNA. In certain embodiments, the nucleic acid is a DNA molecule containing the genome of the DEV defined above.

The nucleic acid may be in free form or in a vector such as a plasmid, BAC, and the like. The nucleic acid may be isolated or contained in the host cell.

Cell Culture The recombinant virus of the invention may be propagated in any competent cell culture. After the required growth of the virus has been achieved, the cells may be separated from the wells using a scraper or trypsin, and the infected cells may be separated from the supernatant by centrifugation.

Examples of competent cells include CEF, embryo-developing eggs, chicken kidney cells, and the like. The cells or virus may be cultured in a culture medium such as Eagle MEM, Reybowitz-L-15 / McCoy5A (1: 1 mixture) culture medium at about 37 ° C. for 3 to 6 days. Infected cells are typically suspended in culture medium containing 10% dimethyl sulfoxide (DMSO) and cryopreserved under liquid nitrogen.

Compositions and Vaccines The present invention also relates to compositions, such as vaccines, comprising one or more DEVs of the invention.

The compositions and vaccines of the invention may contain the DEV in a pharmaceutically or veterinarily acceptable medium or excipient. The compositions and vaccines may further or instead contain the appropriate adjuvant.

Compositions and vaccines according to the present invention may contain suitable solvents such as, for example, aqueous buffers or phosphate buffers. Preferably, the composition and vaccine also include additives such as stabilizers, preservatives, colorants, surfactants, and the like.

For example, the compositions or vaccines of the invention may include one or more additional additives that maintain isotonicity, physiological pH and stability, such as saline (0.85%), phosphate buffered saline (0.85%). PBS), citrate buffer, Tris (hydroxymethylaminomethane (Tris), Tris buffered saline and similar buffers, or may be prescribed with antibiotics such as neomycin or streptomycin, etc. good.

In certain embodiments, the compositions of the invention comprise a preservative.

In another particular embodiment, the composition of the invention comprises a stabilizer.

In another particular embodiment, the composition of the invention comprises an adjuvant. Adjuvants, whether alone or in combination, are sufficient to enhance the immune response, as well as several sources containing a variety of animal-derived proteins and peptides (eg, hormones, cytokines, co-stimulators). It may be obtained from novel nucleic acids derived from the virus and other sources (eg, double-stranded RNA, CpG) and the like.

The compositions of the invention may be liquids (solutions, suspensions, emulsions) or solids (powder, gels, pastes, oils) and can be formulated for any route of administration. Preferably, the compositions of the invention are formulated for injections such as in ovo injections or, for example, for intravenous, subcutaneous, intramuscular, intraorbital, intraocular, intradermal, and / or intraperitoneal injections. To. Alternatively, the compositions of the invention may be formulated for oral, ocular, (eg, by eye drop), nasal, or nasal administration using, for example, aerosols or sprays.

Each vaccine dose may contain an appropriate amount sufficient to elicit a defensive immune response in avian species. Such dose optimization is well known in the art. The amount of antigen per dose may be determined by a known method using an antigen / antibody reaction, for example by an ELISA method.

The vaccines of the invention can be administered as a single dose or in multiple doses, depending on the vaccination protocol.

In certain embodiments, the invention relates to a vaccine comprising a virus, nucleic acid or cell as defined above and a suitable excipient or adjuvant.

In a further specific embodiment, the invention relates to a vaccine comprising a liquid composition of a virus, nucleic acid or cell as defined above and a suitable excipient or adjuvant.

The present invention uses the above viruses, compositions, vaccines, nucleic acids or cells to immunize bird species such as poultry, and immunologically effective amounts of the above viruses, compositions, vaccines, nucleic acids or cells. Further on the method of immunization of avian species by administration.

Further subjects of the invention are birds, in particular poultry, more particularly chickens, more specifically young poultry (3 days after hatching or earlier) or in ovo vaccination or immunization. Concerning the compositions, DEVs, nucleic acids or host cells defined above for use in immunization.

Further subjects of the invention are birds, in particular poultry, more particularly chickens, more specifically young poultry (3 days after hatching or earlier) or in ovo to induce protective immunity. With respect to the compositions, vaccines, DEVs, nucleic acids or host cells defined above for use in.

The present invention relates to methods of vaccination of non-human animals, in particular poultry, more particularly chickens, and more particularly young poultry (3 days after hatching or earlier or in ovo). It also relates to methods comprising administering to said non-human animals the compositions, vaccines, DEVs, nucleic acids or host cells defined above.

A particular subject of the invention is a method of vaccination of poultry, comprising in ovo administration of a composition, vaccine, DEV, nucleic acid or host cell as defined above.

Another particular subject of the invention is a method of vaccination of poultry with administration of the composition, vaccine, DEV, nucleic acid or host cell as defined above on day 1 or 2 after hatching. It is a method to include.

In a further aspect, the invention is a method for inducing an immunogenic or defensive response in a non-human animal against one or more avian pathogens, said non-human animal, in particular poultry, more particularly. To be administered to chickens, and more specifically to young poultry (3 days after hatching or earlier) or in ovo, the compositions, vaccines, DEVs, nucleic acids or host cells defined above. Provide methods including.

As shown in the experimental section, the viruses of the invention are particularly advantageous for vaccination of young poultry (1st, 2nd or 3rd day after hatching) or for in ovo vaccination. In fact, the invention surprisingly reveals that while the viruses of the invention are safe for such early administration, natural or wild-type DEVs are lethal. Such early administration, in combination with the early initiation of immunity caused by these viruses, is particularly advantageous in inducing early protective immunity before poultry can be substantially exposed to the pathogen. ..

In this regard, in a more general aspect, the invention is a method for vaccination or immunization of birds, in particular poultry, and more specifically chickens, wherein the attenuated DEV encoding an antigen. Also related to methods including in ovo administration to birds. The present invention relates to a method for expressing a foreign gene in birds, particularly poultry, and more particularly chickens, comprising in ovo administration of the attenuated DEV containing the foreign gene to the bird. Also involved. The present invention also relates to the use of said DEV containing said gene for expressing a foreign gene in birds by in ovo administration of attenuated DEV. The present invention also relates to said DEV encoding an antigen for use in evoking an immune response or inoculating birds by in ovo administration of attenuated DEV. The DEV preferably contains an inactive endogenous gene, which is attenuated and becomes well tolerated by in ovo injection.

The present invention further relates to a vaccination kit for immunizing the species, including an effective amount of the multivalent vaccine and a means for administering the component to the avian species. For example, such a kit includes a vaccine-filled infusion device according to the invention and instructions for intradermal, subcutaneous, intramuscular, or in ovo injection. Instead, the kit includes a vaccine-filled spray / erosol or eye drop device according to the invention and instructions for use for nasal, oral or mucosal administration.

Further aspects and advantages of the invention will be disclosed in the section of experiments below, which section illustrates the claimed invention.

(Example 1)
Toxicity of wild-type DEV in eggs or young poultry A clinical study was conducted to investigate the pathogenicity or toxicity of DEV in chickens by injection at different time schedules. More specifically, the injection was performed in ovo (3 days before hatching), 1 day after hatching, or 4 days after hatching. The DEV used was a wild-type DEV Jansen strain. The dose was 100 or 1000 pfu / dose. As a control, a PBS solution was administered. Pathogenicity was assessed by measuring mortality each day after hatching.

The results are presented in the table below.

The above results indicate that injection of wtDEV 4 days after hatching is safe and has a 100% survival rate (see group 5). In contrast, 100% of birds died by 4 days after hatching after in-ovo injection of DEV, but in-ovo injection of PBS is safe. Therefore, according to these results, wtDEV is suitable for administration to adults, but surprisingly, wtDEV is lethal when administered to young animals (within 3 days after hatching) or in ovo. It is shown.

(Example 2)
Toxicity of DEVs with the inactive US4 or US5 gene
2.1. Construction of DEVs containing the Inactive US4 or US5 genes US4- or US5-inactive DEVs were constructed in an attempt to reduce toxicity to chicken embryos.

Construction of rpsLneo-DsRed2 cassette
A 2.8 kb DNA fragment of the rpsLneo-DsRed2 cassette was constructed by PCR reaction. Briefly, three PCR reactions were performed. The first PCR reaction used the template of the synthesized fragment of rpsLneo (SEQ ID NO: 3) with SEQ ID NO: 1 (5'-GGCCTGGTGATGATGGCGGGATCGTTGTAT-3') and SEQ ID NO: 2 (5'-CCATGGTGCTGCGCTCAGAAGAACTCGTCA-3'). This was done using a primer pair. The second PCR reaction used the template plasmid of the pSI mammalian expression vector (Promega, Catalog No. E1721) with SEQ ID NO: 4 (5'-ACGAGTTCTTGAGCGCAGCACCATGGCC-3') and SEQ ID NO: 5 (5'-TCGGAGGAGGCCATCCTTAAGAGCTGTAAT-3). ') Primer pair was used. The third PCR reaction used the template plasmid of pIRES2-DsRed2 (Clontech, Catalog No. 632420) with SEQ ID NO: 6 (5'-TACAGCTCTTAAGGATGGCCTCCTCCGAGA-3') and SEQ ID NO: 7 (5'-GCAGTGAAAAAAATGCTTTATTTGTGAAAT-3'). This was done using the primer pair of. Another PCR reaction was performed using a mixture of PCR products from the first and second PCR reactions as a template and SEQ ID NOs: 1 and 5 as primers. This PCR product was mixed with the PCR product from the third PCR reaction and used for the final PCR reaction using the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 7 to obtain the rpsLneo-DsRed2 cassette.

Construction of insertion cassette
DNA fragments of the rpsLneo-DsRed2 cassette with both DEV US4 or US5 region homologous sequences (50 bp each) at both ends of the 5'and 3'ends were constructed by PCR reaction. The PCR reaction was performed using the rpsLneo-DsRed2 cassette as a template. Primer pairs used, US4 inert DEV in SEQ ID NO: 8 (5'-ATGGCAACAATGATAGCTGTGGTGTTAGTTTTTTTGGGACGCGTTTTAGGGGCCTGGTGATGATGGCGGG-3 ') and SEQ ID NO: 9 (5'-TTAAACTAATGGAACGCGTTGGAATTTCAAGTCTTGGCGCCCAAACATCGGCAGTGAAAAAAATGCTTTA-3'), US5 inert DEV in SEQ ID NO: 10 (5'-ATGTATACAGACGTTACGGTCATGTGGGTAGCCGTGATTTTATTTACTATGGCCTGGTGATGATGGCGGG -3') and SEQ ID NO: 11 (5'-TCATACCATACAAAGGCATAGGTACAGCCCACAGGTTAAAAACAAAGAAAGCAGTGAAAAAATGCTTTA-3'). The obtained PCR fragments (US4-rpsLneo-DsRed2 and US5-rpsLneo-DsRed2 cassettes) were electrophoresed and purified.

Construction of recombinant DEV carrying the rpsLneo-DsRed2 gene
Recombinant DEVs carrying the rpsLneo-DsRed2 gene in the US4 or US5 region were constructed by homologous recombination with 0.5 μg US4-rpsLneoDsRed2 or US5-rpsLneoDsRed2-transfected E. coli strains carrying the DEV genome. .. Transfection was performed by electroporation using Gene Pulser XCell (Bio-Rad Laboratories) at 1.75 kV, 25 μF, and 200 ohm. After transfection, E. coli was sown on Luria-Bertani (LB) agar plates and incubated overnight at 30 ° C. E. coli clones carrying the appropriate insert containing the rpsLneo-DsRed2 gene in the US4 or US5 region use a primer pair that amplifies the region (junction 1) between the rpsLneo-DsRed2 gene and the insertion site region of the DEV genome. Identified by. The primers are SEQ ID NO: 12 (5'-AAGTGTATAAATTAGACAAGTAGCTATGCG-3') and SEQ ID NO: 13 (5'-TCAGAAGAACTCGTCAAGAAGGC-3') for the US4 Inactive DEV, and SEQ ID NO: 13 and SEQ ID NO: 14 (5'-) for the US5 Inactive DEV. GTTTATATTGACGCGGAATGTTGAC-3'). DEV DNA was extracted from E. coli clones carrying the appropriate inserts and transfected into CEF using Nucleofector II (Lonza, Basel, Switzerland). Transfected cells include Leibovitz's L-15 (Life Technologies, Catalog No. 41300-39), McCoy's 5A Medium (Life Technologies, Catalog No. 21500-061) (1: 1) and 4% calf serum [ Added to LM (+) medium], sown on 96-well tissue culture plates, then incubated at 37 ° C., 4-5% CO ₂ for 5-7 days until DEV cytopathic effect (CPE) is visible. did. DEVs carrying the rpsLneo-DsRed2 gene in the US4 or US5 region were successfully rescued (DEV / US4 / rpsLneo-DsRed2 or DEV / US5 / rpsLneo-DsRed2).

Verification of Genome Structure The genomic structure of recombinant DEV / US4 / rpsLneo-DsRed2 or DEV / US5 / rpsLneo-DsRed2 amplifies junction regions (junction 1, junction 2, and junction 3) at each end of the inserted gene. It was verified by three PCR reactions. The primer pairs used in the PCR reaction for junction 1 are listed above. In DEV / US4 / rpsLneo-DsRed2, the primer pairs used in the PCR reaction are SEQ ID NO: 6 and SEQ ID NO: 15 (5'-CATTTTAACCGTTTAAGTCAACATTCCGC-3') at Junction 2 and SEQ ID NO: 12 and SEQ ID NO: 15 at Junction 3. .. In DEV / US5 / rpsLneo-DsRed2, the primer pairs used in the PCR reaction are SEQ ID NO: 6 and SEQ ID NO: 16 (5'-ACTGAGATGTTGGACCATCAAATCCTG-3') at junction 2 and SEQ ID NO: 14 and SEQ ID NO: 16 at junction 3. .. The expected size of the PCR product was observed, confirming that DEV / US4 / rpsLneo-DsRed2 and DEV / US5 / rpsLneo-DsRed2 had the expected genomic structure.

2.2. Expression of foreign genes by recombinant DEVs with the Inactive US4 or US5 gene
Expression of the DsRed2 protein by DEV / US4 / rpsLneo-DsRed2 or DEV / US5 / rpsLneo-DsRed2 was confirmed by excitation for DsRed2. Excitation for DsRed2 was performed using CEF infected with DEV / US4 / rpsLneo-DsRed2 or DEV / US5 / rpsLneo-DsRed2. Briefly, 6-well plate CEF cells were infected with DEV / US4 / rpsLneo-DsRed2, DEV / US5 / rpsLneo-DsRed2, or parental DEV strains with an approximately 0.01 multiplicity of infection. Three days after inoculation, cells were excited at 563 nm and red fluorescence was observed in recombinant DEV / US4 / rpsLneo-DsRed2 or DEV / US5 / rpsLneo-DsRed2 plaques, thus confirming actual protein expression by recombinant DEV. rice field.

2.3. Viability and stability of recombinant DEVs with the Inactive US4 or US5 gene
DEV / US4 / rpsLneo-DsRed2 or DEV / US5 / rpsLneo-DsRed2 were passaged 15 times in CEF, confirming the stability of the inserted gene of rpsLneo-DsRed2. The passage was done every 3-4 days. At every 5 passages, DEV / US4 / rpsLneo-DsRed2 or DEV / US5 / rpsLneo-DsRed2 plaques were checked for red fluorescence with a fluorescence microscope and their genomic structure was determined at the junction region using the primers shown in Example 2. Confirmed by PCR analysis to amplify (junction 1, junction 2, and junction 3). Red fluorescence and expected size of the PCR product were observed in all observed viruses, indicating that DEV / US4 / rpsLneo-DsRed2 and DEV / US5 / rpsLneo-DsRed2 carried the rpsLneo-DsRed2 gene for at least 15 passages. It was confirmed.

Toxicity of US4 or US5 Inactive DEV by 2.4 in ovo administration
DEV / US4 / rpsLneo-DsRed2 or DEV / US5 / rpsLneo-DsRed2 was inoculated into day 18 embryos of SPF chickens to investigate their pathogenicity or toxicity to chicken embryos. Chicken embryos were administered approximately 1000 pfu / 0.1 ml of DEV / US4 / rpsLneo-DsRed2, DEV / US5 / rpsLneo-DsRed2, parental DEV, or 0.1 ml of PBS in ovo via a 20 gauge and 1.5 inch needle. Chickens were observed daily for DEV-related clinical signs such as weakness and death for 11 days. The results are shown in the table below.

All chickens in ovo inoculated with the US4 or US5 Inactive DEV died by 4 days after hatching, and 95% of the chickens inoculated with the parent DEV died. The results indicate that the US4 or US5 inactive DEV is still pathogenic and toxic to chicken embryos when administered in ovo.

(Example 3)
Construction of a DEV containing the inert US4 and US5 genes
For the construction of the US4 and US5 Inactive DEVs, a homologous vector was first constructed and then used to generate the virus in E. coli by homologous recombination. Plasmid construction and DNA manipulation were performed essentially according to standard molecular biology techniques (Molecular Cloning: A Laboratory Manual. 4th Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 2012).

Construction of homology vector
A 1.1 kb DNA fragment of the DEV genome adjacent to the US3 and US6 genes was cloned by PCR reaction with the SfiI recognition site added to the insertion site. Briefly, two PCR reactions were performed using DNA extracted from DEV as a template. The primer pairs used were SEQ ID NO: 17 (5'-GCGCATGCTAGCTGATCTAACTTTAC-3'), SEQ ID NO: 18 (5'-GGTGGCCAATAAGGCCTGACGGCAATATGT-3') and SEQ ID NO: 19 (5'-TCAggccttattggccACCAGCTACACAAG-3') and SEQ ID NO: 20 (5'). -GCGAATTCGATTAATTCTCCCGAACTGTTG-3'). Another PCR reaction was performed using a mixture of PCR products from two previous PCR reactions as a template and SEQ ID NOs: 17 and 20 as primers. The obtained PCR fragment was cloned into the pUC18 vector (Genbank Accession No. L09136) after digestion with EcoRI and SphI to obtain pUC18-KAPEVAC-US4US5del-SfiI, which is the US3 and US6 regions of the DEV genome. Including some. Next, a homologous vector containing a promoter derived from the standard loading strain (VP2-STC) and the IBDV VP2 gene was constructed by utilizing the plasmid pUC18-KAPEVAC-US4US5del-SfiI. First, pUC18-KAPEVAC-US4US5del-SfiI was cleaved with SfiI and dephosphorylated with alkaline phosphatase Schwanella species S1B1 recombinant (PAP) (Funakoshi, number DE110). Next, the chicken beta actin (Bac) promoter (SEQ ID NO: 21) and VP2-STC gene were obtained by Bg1I digestion of p45 / 46bacVP2-STC # 11 (US Pat. No. 6,764,684). Finally, this Bac promoter VP2-STC cassette was inserted into the SfiI digestion pUC18-KAPEVAC-US4US5del-SfiI to obtain pUC18-KAPEVAC-US4US5del-BacVP2stc (Fig. 2). This plasmid pUC18-KAPEVAC-US4US5del-BacVP2stc was used to construct DEV / US4US5del / BacVP2stc (Fig. 2).

Building DEV / US4US5del / BacVP2
Construction of a DEV carrying the BacVP2 gene in the US4-US5 region was performed by homologous recombination with an E. coli strain carrying the DEV genome transfected with 0.5 μg of pUC18-KAPEVAC-US4US5del-BacVP2stc. The transfection conditions were described in Example 2. E. coli clones carrying the appropriate inserts containing the BacVP2 gene were identified by PCR using a primer pair that amplifies the region between the BacVP2 gene and the insertion site region of the DEV genome (junction 1, Figure 2). The primers are SEQ ID NO: 22 (5'-GTCCACTATGCCATGACATAGGTG-3') and SEQ ID NO: 23 (5'-GAGCAACTTCGAGCTGATCC-3'). DEV DNA was extracted from E. coli clones carrying the appropriate inserts and transfected into CEF. Transfected cells were incubated until DEV CPE was visible. DEV / US4US5del / BacVP2 has the BacVP2 gene knocked out of its US4 and US5 genes, but was successfully constructed.

Verification of genomic structure
The genomic structure of DEV / US4US5del / BacVP2 was validated by three PCR reactions amplifying junction regions (junction 1, junction 2, and junction 3; Figure 2) at each end of the inserted gene. The primer pairs used in the PCR reaction for junction 1 are listed above. The primer pairs used in the PCR reaction for junction 2 are SEQ ID NO: 24 (5'-GCCAGGGAATCCAGGGAAAAAGAC-3') and SEQ ID NO: 12. In Junction 3, SEQ ID NO: 22 and SEQ ID NO: 12 were used. The expected size of the PCR product was observed, confirming that DEV / US4US5del / BacVP2 had the expected genomic structure.

(Example 4)
Expression of VP2 gene by DEV / US4US5del / BacVP2 Expression of VP2 protein by recombinant DEV / US4US5del / BacVP2 was confirmed by black plaque assay. Briefly, CEF infected with DEV / US4US5del / BacVP2 was immobilized with a methanol: acetone mixture (1: 2) and incubated with anti-IBDV VP2 monoclonal antibody R63 (ATCC #; HB-9490). The VP2 protein was then incubated with a biotinylated anti-mouse IgG antibody (Vector Laboratories, catalog number BA-9200) and then with the VECTASTAIN ABC-AP kit (Vector Laboratories, catalog number AK-5000). The expressed plaques were stained by the addition of NBT / BCIP solution (Roche Applied Science, Catalog No. 1681451). As shown in FIG. 3, expression of the VP2 protein was observed in cells infected with DEV / US4US5del / BacVP2.

(Example 5)
In ovo administration of DEV / US4US5del / BacVP2
DEV / US4US5del / BacVP2 was inoculated into day 18 embryos of SPF chickens. All groups of embryos were vaccinated in ovo with approximately 1000 pfu / 0.1 ml recombinant DEV / US4US5del / BacVP2, parent DEV, or 0.1 ml PBS via 20 gauge and 1.5 inch needles. Chickens were observed daily for DEV-related clinical signs such as weakness and death for 35 days. Five weeks after hatching, chickens were examined for weight gain, autopsied and observed for macroscopically observable lesions. The results are shown in the table below.

The mortality rate of birds that were knocked out for both the US4 and US5 genes and were vaccinated with DEV / US4US5del / BacVP2 was 11.8%, whereas the mortality rate of birds vaccinated with the parent DEV was 100%, and the toxicity and pathogenicity of DEV. Sex has been shown to be substantially reduced by inactivation (eg, deletion) of both the US4 and US5 genes. In addition, the average body weight of surviving birds inoculated with DEV / US4US5del / BacVP2 was comparable to the average body weight of birds inoculated with PBS. From these results, it is possible to demonstrate the effectiveness of the DEV of the present invention for in ovo vaccination.

(Example 6)
Construction of DEV / Coa5 VP2 with Inactive US4 and US5 Genes In this section, we carry the VP2 gene, which has deletions in both the US4 and US5 genes and is driven by the core region of the Bac promoter (Coa5 promoter; SEQ ID NO: 25). Constructed a DEV to do. In the construction of these DEVs, a homologous vector was first constructed and then used to generate a virus in E. coli by homologous recombination.

Construction of pUC18-KAPEVAC-US4US5del
A 1.1 kb DNA fragment of the DEV genome adjacent to the US3 and US6 genes was cloned by PCR reaction (Fig. 4). Briefly, two PCR reactions were performed using DNA extracted from DEV as a template. The primer pairs used were SEQ ID NO: 17 and SEQ ID NO: 26 (5'-GCTTGTGTAGCTGGTTGACGGCAATATG-3') and SEQ ID NO: 27 (5'-CATATTGCCGTCAACCAGCTACACAAGC-3') and SEQ ID NO: 20 (5'-gcGAATTCGATTAATTCTCGAACTGTTG-3'). Another PCR reaction was performed using a mixture of PCR products from two previous PCR reactions as a template and SEQ ID NOs: 17 and 20 as primers. The obtained PCR fragment was cloned into the pUC18 vector after digestion using EcoRI and SphI to obtain pUC18-KAPEVAC-US4US5del (Fig. 4).

Building DEV / US4US5del
Recombinant DEV / US4US5del with deletions in both the US4 and US5 genes was constructed by homologous recombination in E. coli transfected with 0.5 μg pUC18-KAPEVAC-US4US5del and carrying the DEV genome. E. coli clones carrying the appropriate deletion (DH10B / DEV / US4US5del) were identified by PCR using a primer pair that amplifies the region between US3 and US6 (junction 1; Figure 4). The primers used are SEQ ID NO: 22 and SEQ ID NO: 20. DEV DNA was extracted from E. coli clones and transfected into CEF to rescue DEV / US4US5del.

Construction of pUC18-KAPEVAC-UL23del-Coa5VP2
A 1.0 kb DNA fragment of the DEV genome adjacent to the UL24 and UL22 genes was cloned by PCR reaction with the SfiI recognition site added to the insertion site (Fig. 5). Briefly, two PCR reactions were performed using DNA extracted from DEV as a template. The primer pairs used were SEQ ID NO: 28 (5'-GCGCATGCCAATTGTCTAATTCCAG-3'), SEQ ID NO: 29 (5'-CCCGGCCAATAAGGCCACAGAAAAAAGCGCG-3') and SEQ ID NO: 30 (5'-CTGTGGCCTTATTGGCCGGGATCTGGAAC-3') and SEQ ID NO: 31 (5'). -GCGAATTCATGTGCTACGCCCAG-3'). Another PCR reaction was performed using a mixture of PCR products from two previous PCR reactions as a template and SEQ ID NOs: 28 and 31 as primers. The obtained PCR fragment was cloned into the pUC18 vector after digestion using EcoRI and SphI to obtain pUC18-KAPEVAC-UL23del-SfiI. Next, the homology vector containing the promoter and VP2-STC was constructed by utilizing the plasmid pUC18-KAPEVAC-UL23del-SfiI. First, pUC18-KAPEVAC-UL23del-SfiI was cleaved with SfiI and dephosphorylated with PAP. The Coa5 promoter was then obtained from the plasmid pGICOA (US Pat. No. 6,866,852) by BglI and XbaI digestion, and the XbaI-EcoRI fragment (6.3 kb) and EcoRI- of p45 / 46bacVP2-STC # 11 (US Pat. No. 6,764,684). Ligate using a Bg1I fragment (0.1 kb) to obtain p45 / 46COA5VP2-STC # 11. Next, the Coa5 promoter-VP2-STC cassette was excised from p45 / 46COA5VP2-STC # 11 by BglI digestion and ligated with SfiI digestion pUC18-KAPEVAC-UL23del-SfiI to give pUC18-KAPEVAC-UL23del-Coa5VP2. This plasmid was used to construct DEV / US4US5del / UL23 / Coa5VP2.

Construction of pUC18-KAPEVAC-UL26-Coa5VP2
A 1.0 kb DNA fragment of the DEV genome adjacent to the UL26 and UL27 genes was cloned by PCR reaction with the SfiI recognition site added to the insertion site (Fig. 6). Briefly, two PCR reactions were performed using DNA extracted from DEV as a template. The primer pairs used were SEQ ID NO: 32 (5'-CGGTCGACACTCCCAGGGGTGAAGC-3'), SEQ ID NO: 33 (5'-CGGCCAATAAGGCCAAGAATGCATTCGGCC-3') and SEQ ID NO: 34 (5'-TGGCCTTATTGGCCGCCGTATGAATTGCGC-3') and SEQ ID NO: 35 (5'). -GCGAGCTCCTGCAACCACAGACCGC-3'). Another PCR reaction was performed using a mixture of PCR products from two previous PCR reactions as a template and SEQ ID NOs: 32 and 35 as primers. The obtained PCR fragment was cloned into the pUC18 vector after digestion with SalI and SacI to obtain pUC18-KAPEVAC-UL26-SfiI. Next, a homologous vector containing a promoter derived from a standard loading strain and the IBDV VP2 gene was constructed by utilizing the plasmid pUC18-KAPEVAC-UL26-SfiI. First, pUC18-KAPEVAC-UL26-SfiI was cleaved with SfiI and dephosphorylated with PAP. The Coa5 promoter-VP2-STC cassette was excised from pUC18-KAPEVAC-UL23del-Coa5VP2 by SfiI digestion and ligated with SfiI digestion pUC18-KAPEVAC-UL26-SfiI to give pUC18-KAPEVAC-UL26-Coa5VP2. This plasmid was used to construct DEV / US4US5del / UL26 / Coa5VP2.

Construction of pUC18-KAPEVAC-UL45-Coa5VP2
A 1.0 kb DNA fragment of the DEV genome adjacent to the UL45 and UL46 genes was cloned by PCR reaction with the SfiI recognition site added to the insertion site (Fig. 7). Briefly, two PCR reactions were performed using DNA extracted from DEV as a template. The primer pairs used were SEQ ID NO: 36 (5'-CGGTCGACATAGAACGCGCTTCATCTAA-3') and SEQ ID NO: 37 (5'-TGGCCAATAAGGCCGTTTATTGTTTATTAT-3') and SEQ ID NO: 38 (5'-CGGCCTTATTGGCCAATCTGATTCATCCAA-3') and SEQ ID NO: 39 (5'). -GCGAGCTCCGCCTAATCACAATCGGTATTG-3'). Another PCR reaction was performed using a mixture of PCR products from two previous PCR reactions as a template and SEQ ID NOs: 36 and 39 as primers. The obtained PCR fragment was cloned into the pUC18 vector after digestion with SalI and SacI to obtain pUC18-KAPEVAC-UL45-SfiI. Next, a homologous vector containing a promoter derived from a standard loading strain and the IBDV VP2 gene was constructed by utilizing the plasmid pUC18-KAPEVAC-UL45-SfiI. First, pUC18-KAPEVAC-UL45-SfiI was cleaved with SfiI and dephosphorylated with PAP. The Coa5 promoter-VP2-STC cassette was excised from pUC18-KAPEVAC-UL23del-Coa5VP2 by SfiI digestion and ligated with SfiI digestion pUC18-KAPEVAC-UL45-SfiI to give pUC18-KAPEVAC-UL45-Coa5VP2. This plasmid was used to construct DEV / US4US5del / UL45 / Coa5VP2.

Construction of pUC18-KAPEVAC-UL50-Coa5VP2
A 1.0 kb DNA fragment of the DEV genome adjacent to the UL50 and UL51 genes was cloned by PCR reaction with the SfiI recognition site added to the insertion site (Fig. 8). Briefly, two PCR reactions were performed using DNA extracted from DEV as a template. The primer pairs used were SEQ ID NO: 40 (5'-CCGCATGCGCAACTATATATGTCGGTC-3') and SEQ ID NO: 41 (5'-GGGCCAATAAGGCCCAAAAGTACATTTTGT-3') and SEQ ID NO: 42 (5'-GGGCCTTATTGGCCCAATTTATTTACTATT-3') and SEQ ID NO: 43 (5'). -GCGAATTCTGGATATGATATACCGTTGC-3'). Another PCR reaction was performed using a mixture of PCR products from two previous PCR reactions as a template and SEQ ID NOs: 40 and 43 as primers. The obtained PCR fragment was cloned into the pUC18 vector after digestion using EcoRI and SphI to obtain pUC18-KAPEVAC-UL50-SfiI. Next, a homologous vector containing a promoter derived from a standard loading strain and the IBDV VP2 gene was constructed by utilizing the plasmid pUC18-KAPEVAC-UL50-SfiI. First, pUC18-KAPEVAC-UL50-SfiI was cleaved with SfiI and dephosphorylated with PAP. The Coa5 promoter-VP2-STC cassette was excised from pUC18-KAPEVAC-UL23del-Coa5VP2 by SfiI digestion and ligated with SfiI digestion pUC18-KAPEVAC-UL50-SfiI to give pUC18-KAPEVAC-UL50-Coa5VP2. This plasmid was used to construct DEV / US4US5del / UL50 / Coa5VP2.

Construction of DEV / US4US5del / Coa5VP2stc
Construction of recombinant DEVs with deletions in the US4 and US5 genes and carrying the Coa5VP2 gene in the UL23, UL26 / UL27, UL45 / UL46, or UL50 / UL51 regions can be performed with DEV / US4US5del and pUC18-KAPEVAC-UL23del-Coa5VP2, Homologous recombination was performed on 0.5 μg of one of pUC18-KAPEVAC-UL26-Coa5VP2, pUC18-KAPEVAC-UL45-Coa5VP2, or pUC18-KAPEVAC-UL50-Coa5VP2. The transfection conditions are described in Example 2. After transfection, E. coli clones carrying the appropriate insert containing the Coa5VP2 gene use a primer pair that amplifies the region between the Coa5VP2 gene and the insertion site region of the DEV genome (junction 1, FIGS. 5-8). Identified by PCR. The primers are SEQ ID NO: 23 and SEQ ID NO: 28 at the insertion site UL23, SEQ ID NO: 32 at the insertion site UL26 / UL27, SEQ ID NO: 36 at the insertion site UL45 / UL46, or SEQ ID NO: 40 at the insertion site UL50 / UL51. Modified DEV DNA was extracted from E. coli clones carrying the appropriate inserts and transfected into CEF using Nucleofector II. Transfected cells were added to LM (+) medium, sown on 96-well tissue culture plates, and then incubated at 37 ° C., 4-5% CO ₂ for 5-7 days until DEV CPE became visible. After transfection, DEVs carrying the Inactive US4 and US5 genes and the Coa5VP2 gene were successfully rescued (DEV / US4US5del / UL23 / Coa5VP2, DEV / US4US5del / UL26 / Coa5VP2, DEV / US4US5del / UL45 / Coa5VP2, And DEV / US4US5del / UL50 / Coa5VP2).

Verification of genomic structure
The genomic structures of DEV / US4US5del / UL23 / Coa5VP2, DEV / US4US5del / UL26 / Coa5VP2, DEV / US4US5del / UL45 / Coa5VP2, and DEV / US4US5del / UL50 / Coa5VP2 are junctions at each end of the inserted gene. 1, Junction 2, and Junction 3; were verified by three PCR reactions that amplify (Figs. 5 to 8). The primer pairs used in the PCR reaction for junction 1 are listed above. The primer pairs used in the PCR reaction for junction 2 are SEQ ID NO: 24 and SEQ ID NO: 31 (insertion site UL23), SEQ ID NO: 35 (insertion site UL26 / UL27), SEQ ID NO: 39 (insertion site UL45 / UL46), or sequence. It is number 43 (insertion site UL50 / UL51). At junction 3, primer pair SEQ ID NO: 28 / SEQ ID NO: 31 (insertion site UL23), SEQ ID NO: 32 / SEQ ID NO: 35 (insertion site UL26 / UL27), SEQ ID NO: 36 / SEQ ID NO: 39 (insertion site UL45 / UL46), or SEQ ID NO: 40 / SEQ ID NO: 43 (insertion site UL50 / UL51) was used. Expected size is observed for PCR products, and genomic structures expected to be DEV / US4US5del / UL23 / Coa5VP2, DEV / US4US5del / UL26 / Coa5VP2, DEV / US4US5del / UL45 / Coa5VP2, and DEV / US4US5del / UL50 / Coa5VP2. It was confirmed that it had.

(Example 7)
Expression of the VP2 gene by DEV / Coa5 VP2 containing the inactive US4 and US5 genes
Expression of VP2 protein by DEV / US4US5del / UL23 / Coa5VP2, DEV / US4US5del / UL26 / Coa5VP2, DEV / US4US5del / UL45 / Coa5VP2, and DEV / US4US5del / UL50 / Coa5VP2 was confirmed by black plaque assay and Western blot analysis. The method of black plaque assay is described in Example 4. As shown in FIG. 9, expression of the VP2 protein infected DEV / US4US5del / UL23 / Coa5VP2, DEV / US4US5del / UL26 / Coa5VP2, DEV / US4US5del / UL45 / Coa5VP2, or DEV / US4US5del / UL50 / Coa5VP2. Observed in cells. Western blots use CEF and anti-IBDV VP2 monoclonal antibody R63 infected with DEV / US4US5del / UL23 / Coa5VP2, DEV / US4US5del / UL26 / Coa5VP2, DEV / US4US5del / UL45 / Coa5VP2, or DEV / US4US5del / UL50 / Coa5VP2. I went there. Briefly, CEF in a 12-well plate was infected with a recombinant virus or one of the parent DEVs. Four days after inoculation, cells were harvested with trypsin and centrifuged at 913 xg for 5 minutes. The pellet was washed with PBS and resuspended in 25 μl PBS. After adding the same volume of 2 × SDS sample buffer (130 mM Tris-Cl (pH 6.8), 6% SDS, 20% glycerol, 10% 2-mercaptoethanol and 0.01% bromophenol blue), cells The suspension was boiled for 5 minutes. Samples were separated by SDS-PAGE using 12.5% polyacrylamide gel and transferred to PVDF membrane (Immobilon-P, Millipore). Membranes were completely dried and then incubated with R63 monoclonal antibody. After flushing the R63 antibody, use the biotinylated anti-mouse IgG antibody and then the VECTASTAIN ABC-AP kit. The protein bound to the R63 monoclonal antibody was visualized by the addition of NBT / BCIP solution. The 40-kilodalton protein band, which is the expected size of VP2 protein, was observed in all lanes with recombinant cells (Figure 10), confirming that recombinant virus-infected cells express VP2 protein. Was done.

(Example 9)
Stability of DEV / Coa5VP2 containing the inert US4 and US5 genes
DEV / US4US5del / UL23 / Coa5VP2, DEV / US4US5del / UL26 / Coa5VP2, DEV / US4US5del / UL45 / Coa5VP2, and DEV / US4US5del / UL50 / Coa5VP2 were passaged 15 times in CEF and the stability of the BacVP2 inserted gene. Was confirmed correctly. The passage was done every 3 to 4 days. For every 5 passages, cells infected with DEV / US4US5del / UL23 / Coa5VP2, DEV / US4US5del / UL26 / Coa5VP2, DEV / US4US5del / UL45 / Coa5VP2, or DEV / US4US5del / UL50 / Coa5VP2 are black for VP2 gene expression. Investigate by plaque assay, its genomic structure is confirmed by PCR analysis to amplify the junction regions (junction 1, junction 2, and junction 3; FIGS. 5-8) and the primers are shown in Example 7. As a result, neither reversion mutations of the US4 and US5 genes nor deletions on the Coa5VP2 gene were observed, indicating that these viruses are stable in CEF.

(Example 10)
In ovo administration of DEV / Coa5VP2 containing the inert US4 and US5 genes In this study, we will investigate the in ovo safety and defense efficiency against toxic IBDV.

Inoculate DEV / US4US5del / UL23 / Coa5VP2, DEV / US4US5del / UL26 / Coa5VP2, DEV / US4US5del / UL45 / Coa5VP2, and DEV / US4US5del / UL50 / Coa5VP2 or parent DEV into day 18 embryos of SPF chickens. All groups of embryos were vaccinated in ovo with approximately 1000 pfu / 0.1 ml recombinant virus, parental DEV, or 0.1 ml PBS via 20 gauge and 1.5 inch needles. Chickens are observed daily for DEV-related clinical signs such as weakness and death for 42 days. Chickens were weighed weekly for 1 to 5 weeks of age to assess humoral immunity to IBDV and to determine viral toxicity. Anti-IBDV antibodies are quantified using a commercially available IBDV ELISA kit (ID SCREEN IBD VP2; ID Vet). All chickens except Group 1 are loaded with a 10 ³ mean embryonic infectious titer (EID ₅₀ ) of the toxic IBDV standard load (STC) strain via the oral route. Chickens are observed daily for IBD-related clinical signs such as weakness and death. Seven days after loading, the chicken is autopsied and observed for macroscopically observable Bursa lesions such as edema, discoloration, atrophy, hemorrhage, and yellow or gelatinous exudate. At autopsy, body weight and bursa were also measured to calculate the B / B index, which is the ratio of bursa weight to loaded bird weight divided by the same ratio of unloaded birds.

The results of this study can confirm in vivo safety, stability, and effective expression.

Claims (29)

Duck plague virus (DEV) with the inactive US4 and US5 genes. The DEV according to claim 1, wherein the US4 and US5 genes are mutated, deleted, or interrupted independently of each other. The DEV according to claim 1 or 2, wherein at least 20% of the respective coding sequences of the US4 and US5 genes are deleted. DEV according to any one of claims 1 to 3, wherein the intergenic region between the US4 and US5 genes is deleted. 13. DEV. The DEV according to any one of claims 1 to 5, further comprising the inert UL23, US7 and / or UL4 genes. (i) Deletion of nucleotide regions containing at least 50% of the US4 gene, all of the intergenic region between the US4 and US5 genes, and at least 50% of the US5 gene, and (ii) at least 50% of the UL23 gene sequence. DEV according to claim 6, comprising a deletion. (i) Deletion of nucleotide regions containing at least 50% of the US4 gene, all of the intergenic region between the US4 and US5 genes, and at least 50% of the US5 gene, and (ii) at least 50% of the US7 gene sequence. DEV according to claim 6, comprising a deletion. (i) Deletion of nucleotide regions containing at least 50% of the US4 gene, all of the intergenic region between the US4 and US5 genes, and at least 50% of the US5 gene, and (ii) at least 50% of the UL4 gene sequence. DEV according to claim 6, comprising a deletion. The DEV according to any one of claims 1 to 9, further comprising a foreign nucleic acid. The DEV according to claim 10, wherein the foreign nucleic acid is located on the inactive US4 or US5 gene. The virus contains a deletion of a nucleotide region containing at least 50% of the US4 gene, all of the intergenic region between the US4 and US5 genes, and at least 50% of the US5 gene, and the region in which the foreign nucleic acid is deleted. DEV according to claim 11, which is located on behalf of. The foreign nucleic acid is selected from the UL4 gene, UL44 gene, UL27-UL26 intergenic region, UL23 gene, UL45-UL46 intergenic region, UL50-UL51 intergenic region, US7 gene, US7-US8 intergenic region, or US10 gene. DEV according to claim 10, which is located at the insertion site. The DEV according to any one of claims 10 to 13, wherein the foreign nucleic acid encodes an antigen or an immunostimulatory molecule. DEV according to claim 14, wherein the foreign nucleic acid encodes an antigen derived from an avian pathogen. Antigens are avian paramyxovirus type 1 antigen protein or peptide, Newcastle disease virus (NDV) F protein or fragment thereof, Gamboro disease virus antigen peptide, infectious Fabricius sac virus (IBDV) VP2 protein or its fragments. Fragments, infectious laryngeal tracheitis virus (ILTV) antigenic peptide, gB protein or fragment thereof, mycoplasma galicepticum antigen peptide, 40K protein or fragment thereof, and bird influenza virus antigen peptide, surface protein hemaglutinine (HA) or its fragment. DEV according to claim 14 or 15, which is a fragment. DEV according to claim 16, wherein the antigenic peptide is the VP2 protein of IBDV or an immunogenic fragment thereof, or the hemagglutinin (HA) protein of influenza virus or an immunogenic fragment thereof. A nucleic acid molecule comprising the genome of the DEV according to any one of claims 1 to 17. A host cell comprising the DEV according to any one of claims 1 to 17 or the nucleic acid molecule according to claim 18. A method for producing or replicating the DEV according to any one of claims 1 to 17, wherein the competent cell is infected with the nucleic acid molecule according to claim 18 or the DEV according to claim 1. Methods involving collecting DEVs. DEV according to any one of claims 1 to 17, or nucleic acid according to claim 18, for use in vaccination or immunization of poultry. DEV according to any one of claims 1 to 17, or nucleic acid according to claim 18, for use in vaccination or immunization of chickens. DEV according to any one of claims 1 to 17, or nucleic acid according to claim 18, for use in inducing defense immunity in poultry. DEV according to any one of claims 1 to 17, or nucleic acid according to claim 18, for use in inducing defense immunity in chickens. The DEV according to any one of claims 2 1 to 24, which is administered by injection. The D EV according to any one of claims 21 to 25, which is administered in ovo or on the 1st or 2nd day after hatching. The DEV according to any one of claims 1 to 17, the nucleic acid according to claim 18, or the host cell according to claim 19, and a pharmaceutically or veterinarily acceptable excipient or carrier. Composition containing. 27. The composition of claim 27, further comprising an adjuvant. A vaccination kit for immunizing birds with the following ingredients:
The composition according to claim 27 or 28 in an effective amount, and
b. A kit comprising means for administering the composition to the birds.

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